Zhou Dan, Tang Weiwei, Zhang Yun, An Han-Xiang
Department of Medical Oncology, Xiang'an Hospital of Xiamen University, Xiamen, Fujian, China,
Key Laboratory of Design and Assembly of Functional Nanostructures, Fujian Provincial Key Laboratory of Nanomaterials, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China,
Cancer Manag Res. 2019 Mar 27;11:2457-2470. doi: 10.2147/CMAR.S189937. eCollection 2019.
JAM3, an adhesion and transmigration regulatory element, is abundantly expressed in intestinal epithelial cells. However, its expression and function in colorectal cancer (CRC) remain unknown. In this study, we explored its epigenetic mechanism and biological role in CRC.
Bioinformatics analysis was used to analyze the expression and methylation level of JAM3 in CRC. Methylation and expression status of JAM3 were then validated by quantitative methylation-specific PCR (qMSP) and quantitative PCR in tissues, plasma samples, and cell lines. Flow cytometry, Western blot, transwell, siRNA, colony formation, and transfection were used to evaluate the biological function of JAM3.
We initially found that JAM3 was frequently methylated and downregulated in CRC based on bioinformatics tools. qMSP validation showed that the methylation levels of JAM3 were increased in 75% (18/24) of CRC tissues, 61% (11/18) plasma samples, and all four CRC cell lines and were significantly associated with tumor stage in CRC tissues. Moreover, JAM3 was downregulated in primary CRC tissues, plasma samples, and CRC cell lines as compared with that in nonmalignant controls, although its expression could be recovered after demethylation treatment. Restoration of JAM3 repressed CRC cell viability, colony formation, and migration. In addition, siRNA-mediated depletion of JAM3 in NCM460 cells improved the clonogenicity and migration capability, whereas it suppressed cell apoptosis and cell-cycle arrest. These functional effects were accompanied with alterations of several epithelial cell markers, including E-cadherin, vimentin, phosphor-β-catenin (ser552), and TJP1, which were responsible for epithelial-mesenchymal transition.
The findings indicated that JAM3 may be a novel tumor suppressor gene with epigenetic reduction in CRC and can be used as a potential noninvasive biomarker for CRC diagnosis.
JAM3是一种黏附与迁移调节元件，在肠道上皮细胞中大量表达。然而，其在结直肠癌（CRC）中的表达及功能尚不清楚。在本研究中，我们探讨了其在CRC中的表观遗传机制及生物学作用。
采用生物信息学分析CRC中JAM3的表达及甲基化水平。然后通过定量甲基化特异性PCR（qMSP）及定量PCR在组织、血浆样本和细胞系中验证JAM3的甲基化及表达状态。运用流式细胞术、蛋白质免疫印迹法、Transwell实验、小干扰RNA（siRNA）、集落形成实验及转染实验评估JAM3的生物学功能。
基于生物信息学工具，我们最初发现CRC中JAM3经常发生甲基化且表达下调。qMSP验证显示，75%（18/24）的CRC组织、61%（11/18）的血浆样本以及所有四种CRC细胞系中JAM3的甲基化水平均升高，且与CRC组织中的肿瘤分期显著相关。此外，与非恶性对照相比，原发性CRC组织、血浆样本及CRC细胞系中JAM3表达下调，不过去甲基化处理后其表达可恢复。JAM3的恢复抑制了CRC细胞的活力、集落形成及迁移。此外，siRNA介导的NCM460细胞中JAM3的缺失提高了克隆形成能力及迁移能力，而抑制了细胞凋亡及细胞周期停滞。这些功能效应伴随着几种上皮细胞标志物的改变，包括E-钙黏蛋白、波形蛋白、磷酸化β-连环蛋白（ser552）和紧密连接蛋白1，它们与上皮-间质转化有关。
研究结果表明，JAM3可能是一种在CRC中因表观遗传而表达降低的新型肿瘤抑制基因，可作为CRC诊断的潜在非侵入性生物标志物。
